1. Field of the Invention
The present invention relates to a method for the isolation of octopine and its analogues from diastereoisomers of these compounds.
2. Description of the Prior Art
Considerable activity exists in the area of genetic engineering of microorganisms. However, only recently has there been significant activity involving genetic engineering of higher plants. One method which has been proposed for introducing genetic material into higher plants involves the use of the bacterium Agrobacterium tumefaciens to introduce a crown gall tumor in a dicotyledoneous plant. Variant A. tumefaciens bacterial strains contain a large Ti (tumor-inciting) plasmid, part of which, a specific segment called the T-DNA (transferred DNA), integrates into the plant nuclear DNA where it is retained and expressed even after the tumors redifferientiate. Accordingly, the Ti plasmid is a possible vector for accomplishing genetic engineering in plants. See, Ream & Gordon, Crown Gall Disease and Prospects for Genetic Manipulation of Plants", Science 218:854-859 (1982).
Crown gall tumor cells produce opines, which are unusual amino acid derivatives not found in normal plant cells. The ability of transformed cells to synthesize these amino acid derivatives depends strictly on the bacterial strain which causes the tumor. Furthermore, bacteria which induce a specific amino acid derivative can utilize that derivative as a single source of carbon and nitrogen, but cannot utilize opines produced by tumors caused by other strains of bacteria. Accordingly, opines can be utilized in the preparation of bacterial growth media useful for the selection of appropriate strains of bacteria. Other derivatives are toxic to the bacteria containing the appropriate catabolic enzymes coded with the Ti plasmid. See Petit & Tempe, "Isolation of Agrobacterium Ti-Plasmid Regulatory Mutants", Molec. Gen. Genet. 167:147-155 (1978). Use of these derivatives and selected media permits the selection of mutant strains of bacteria which do not have the ability to catabolize the toxic derivatives.
Accordingly, a source of opines useful for producing the selective media is needed. While it is possible to isolate different opines from crown gall tumors of plants, the isolation techniques are tedious and a general synthetic method capable of synthesizing different opines in good yield is needed. See, Firmin & Fenwick, Phytochemistry 16:761-762 (1977).
Synthetic procedures for the synthesis of opines and particularly for the synthesis of D-octopine and its derivatives exist in the prior art but suffer from disadvantages caused by the difficulty of separating D-octopine (or an analogue of D-octopine) from the diastereoisomer produced by the synthetic method, known as L-allooctopine (or the corresponding L-allooctopine analogue).
The aforementioned Petit and Tempe, disclose a chemical synthesis of octopine and analogues based on the general method of Izumiya et al, J. Amer. Chem. Soc. 79:652-658 (1957) involving condensation of an amino acid with the suitable bromo derivative of propionic acid in the presence of barium hydroxide.
Goto et al, Bull. Chem. Soc. Jpn. 55:261-265 (1982), prepare different octopine isomers by reacting the corresponding alanine derivative and 2-Br-5-acetamidopentanoic acid followed by deacetylation and guanidination with an overall yield of 61%. In both these references, precipitation of the octopine isomers was accomplished from a water-ethanol mixture.
Biellman et al, Bioorganic Chemistry 6:89-93 (1977), synthesize the present compounds by reductive deamination with cyanoborohydride with a yield of between 28 and 38%. The compounds were further purified by chromatography on a cationic exchange substrate and the diastereoisomers were separated by fractional crystallization from a water-ethanol mixture.
Moore and Wilson, J. Bio. Chem. 119:573-83 (1937) obtain octopine by aqueous extraction from scallop muscle. After separation from glycogen and protein and a series of various other treatments the resulting solution was concentrated at 40.degree.-50.degree. C. and octopine was allowed to crystallize for several days at 0.degree.-4.degree. C. The product was dissolved in hot water and re-crystallized from an alcohol-water mixture (80%).
Goto et al, also disclose the chemical synthesis of D-octopinic acid as an intermediate in the synthesis of the octopine isomers (see above). The octopinic acid isomers were obtained from alanine and 2-Br-5-acetamidopentanoic acid in the presence of barium hydroxide with a yield of 16%.
Accordingly, an improved method for the separation of octopine and its analogues from their diastereoisomers is necessary in order for these synthetic methods to be fully developed.